Human induced pluripotent stem cell based hepatic-modeling of lipid metabolism associated TM6SF2 E167K variant.

BACKGROUND AND AIMS: TM6SF2 rs58542926 (E167K) is related to increased prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD). Conflicting mouse study results highlight the need for a human model to understand this mutation's impact. This study aims to create and characterize a reliable human in vitro model to mimic the effects of the TM6SF2-E167K mutation for future studies. APPROACH AND RESULTS: We used gene editing on human human-induced pluripotent stem cells (iPSC) from a healthy individual to create cells with the TM6SF2-E167K mutation. After hepatocyte directed differentiation, we observed decreased TM6SF2 protein expression, increased intracellular lipid droplets and total cholesterol in addition to reduced VLDL secretion. Transcriptomics revealed upregulation of genes involved in lipid, fatty acid, and cholesterol transport, flux, and oxidation. Global lipidomics showed increased lipid classes associated with ER stress, mitochondrial dysfunction, apoptosis, and lipid metabolism. Additionally, the TM6SF2-E167K mutation conferred a pro-inflammatory phenotype with signs of mitochondria and ER stress. Importantly, by facilitating protein folding within the ER of hepatocytes carrying TM6SF2-E167K mutation, VLDL secretion and ER stress markers improved. CONCLUSIONS: Our findings indicate that induced hepatocytes generated from iPSCs carrying the TM6SF2-E167K recapitulate the effects observed in human hepatocytes from individuals with the TM6SF2 mutation. This study characterizes an in vitro model that can be used as a platform to identify potential clinical targets and highlights the therapeutic potential of targeting protein misfolding to alleviate ER stress and mitigate the detrimental effects of the TM6SF2-E167K mutation on hepatic lipid metabolism.

Combining descriptive and predictive modeling to systematically design depth filtration-based harvest processes for biologics.

Advances in upstream production of biologics-particularly intensified fed-batch processes beyond 10% cell solids-have severely strained harvest operations, especially depth filtration. Bioreactors containing high amounts of cell debris (more than 40% particles <10 µm in diameter) are increasingly common and drive the need for more robust depth filtration processes, while accelerated timelines emphasize the need for predictive tools to accelerate development. Both needs are constrained by the current limited mechanistic understanding of the harvest filter-feedstream system. Historically, process development relied on screening scale-down depth filter devices and conditions to define throughput before fouling, indicated by increasing differential pressure and/or particle breakthrough (measured via turbidity). This approach is straightforward, but resource-intensive, and its results are inherently limited by the variability of the feedstream. Semi-empirical models have been developed from first principles to describe various mechanisms of filter fouling, that is, pore constriction, pore blocking, and/or surface deposit. Fitting these models to experimental data can assist in identifying the dominant fouling mechanism. Still, this approach sees limited application to guide process development, as it is descriptive, not predictive. To address this gap, we developed a hybrid modeling approach. Leveraging historical bench scale filtration process data, we built a partial least squares regression model to predict particle breakthrough from filter and feedstream attributes, and leveraged the model to demonstrate prediction of filter performance a priori. The fouling models are used to interpret and provide physical meaning to these computational models. This hybrid approach-combining the mechanistic insights of fouling models and the predictive capability of computational models-was used to establish a robust platform strategy for depth filtration of Chinese hamster ovary cell cultures. As new data continues to teach the computational models, in silico tools will become an essential part of harvest process development by enabling prospective experimental design, reducing total experimental load, and accelerating development under strict timelines.

Extracellular vesicles as mediators of in vitro neutrophil swarming on a large-scale microparticle array.

Neutrophils combat infections and promote healing of damaged tissues while protecting the surrounding healthy tissue through a process called swarming. Swarming neutrophils release soluble factors that recruit additional neutrophils and shape the inflammation response. Additionally, neutrophils release extracellular vesicles (EVs), which are gaining attention as important intercellular mediators. We developed a large-scale array of bioparticles on a glass substrate that triggers neutrophil swarming in vitro in a spatially and temporally controlled manner that facilitates the analysis of neutrophil migration. Our platform can generate 30 000 neutrophil swarms on a glass slide in a highly reproducible manner (98% patterning efficiency), which produces an EV-rich supernatant that enables quantitative characterization of inflammation-specific EVs. Healthy neutrophils were able to form uniform swarms across the bioparticle array, which demonstrates a high degree of intercellular coordination. However, neutrophils swarming on the bioparticle array tended to have a lower radial velocity than neutrophils swarming toward a single target. After collecting and isolating EVs released by swarming and non-swarming neutrophils, we found that neutrophils constitutively release exosomes and microvesicles. Furthermore, EVs released by swarming neutrophils cause neutrophil activation and contain the proinflammatory mediator galectin-3, suggesting that EVs have an active role during neutrophil swarming. Ultimately, understanding EVs' role in intercellular communication during swarming will improve understanding of the complex signaling pathways involved in the regulation of inflammation.